3.1 Characterization of CDH and CHMO
CDH and CHMO were cloned separately into the pSEVA244_T vector[18] to characterize their in vivoactivity. As CDH catalyzes an equilibrium reaction, the kinetic parameters were assayed for both reaction directions (Table 1). For the reverse reaction with cyclohexanone as substrate, CDH showed a 10 times lower Vmax compared to the forward reaction. On the other hand, the KS values differed by a factor of almost 100 in favor of the reverse reaction (0.05 and 3.57 mM for cyclohexanol and cyclohexanone, respectively). Furthermore, we theoretically and experimentally assessed the cyclohexanol/cyclohexanone concentration ratio at equilibrium. Utilizing the group contribution method[24] assuming a physiological intracellular NADH to NAD concentration ratio of 10.6 under aerobic conditions[25], this ratio was determined to be 1.9 (Supplementary Information, Section 4). It was confirmed experimentally by applying different initial alcohol and ketone concentrations giving a cyclohexanol/cyclohexanone concentration ratio of 1.95 ± 0.29 after 16 h (Figure S2). This thermodynamic preference of the backward reaction, together with the low KS value for cyclohexanone emphasizes the necessity of an efficient cyclohexanone withdrawal by the successive enzyme in the cascade, i.e., CHMO.
Substantial research has been conducted with a cyclohexanone monooxygenase originating from Acinetobacter sp.[26]. Generally, the substrate as well as product toxicity, are features of Baeyer-Villiger monooxygenase-catalyzed reactions [22, 23]. Substrate toxicity was generally observed at aqueous concentrations in the mM-range, which should thus be avoided during the cascade reaction. Furthermore, CHMO may be inhibited by the cascade intermediate cyclohexanol and its product ε-CL. Acidovorax CHMO indeed was found to be highly prone to inhibition by cyclohexanol (Figure 2A). At a cyclohexanol concentration as low as 0.4 mM, the high initial CHMO activity of 160.3 ± 0.1 U gCDW-1 was found to be reduced to half this rate. Cyclohexanol concentrations ≥ 1.7 mM completely abolished CHMO activity. However, up to an ε-CL concentration of 17 mM, no product inhibition was found for CHMO (Figure 2B).
Summing up, these results emphasize that the produced cyclohexanol needs to be directly converted by CDH to avoid CHMO inhibition. High intracellular CDH and CHMO activities are important to avoid any accumulation of alcohol and ketone intermediates, as already low alcohol amounts can be expected to inherently reinforce such accumulation.